Currently, the molecular events in skeletal muscle that underlie exercise-induced fatigue, changes during disuse, and the symptoms of diseases such as myotonia congenita are not fully understood. For example, in disuse experiments, the chloride permeability of muscle has been shown to increase as a result of reduced protein kinase C activity, but the signaling events that induce those changes are unknown. The long- term goal of the research in this proposal is to elucidate physiological and pathophysiological mechanisms of muscle adaptation. This will be achieved by examining a novel purinergic signaling cascade discovered by the PI. The discovery of this signaling cascade was surprising, as it had been known since 1969 that human skeletal muscle releases ATP during exercise. The mechanism likely went undetected because it is unique to mammalian muscle relative to amphibian and most of the previous examinations used amphibian muscle or cell culture preparations. The recent study by the PI revealed that physiologically relevant levels of extracellular ATP act on P2Y1 receptors to rapidly (seconds to minutes) inhibit chloride channels in mammalian skeletal muscle. Because chloride channels are responsible for most of the resting conductance in skeletal muscle, this discovery has significant implications for the physiology of muscle excitability and fatigue. Pathologically, mutations in ClC-1, by far the predominant muscle chloride channel and the likely target of P2Y1 receptors, underlie the hyperexcitability seen in patients with Thomsen and Becker myotonias. Moreover, recent reports suggest that muscle chloride channels regulate the onset of exercise-related fatigue. The aims of the research in this proposal are to characterize further the P2Y1/chloride channel signaling mechanism and to determine the effects of this cascade on active electrical properties in muscle. Electrophysiological, biochemical and pharmacological techniques will be used in Specific Aims 1 & 2 to determine whether ClC-1 and protein kinase C, a known regulator of ClC-1, function in the P2Y1/chloride channel pathway. Optical and electrophysiological methods will be used in Specific Aim 3 to measure the effects of chloride channel inhibition by P2Y1 receptors on the propagation of action potentials in the sarcolemma and transverse tubular system. By providing mechanistic insights and examining the physiological role of a novel purinergic signaling cascade, the results from the proposed studies will have implications for exercise-related muscle fatigue, muscle disuse, and disorders such as myotonia congenita. PUBLIC HEALTH RELEVANCE: The focus of this proposal is to examine a novel cell signaling cascade in mammalian skeletal muscle that is activated by extracellular ATP and results in the inhibition of chloride channels. The proposed research aims to identify key signaling molecules that participate in the mechanism and to determine the role of this signaling cascade in active muscle. This research will provide insights into the physiological role of ATP release during muscle activity and it has implications for exercise-induced fatigue, muscle disuse, and treating disorders such as myotonia congenita.